Desiccation, Dormancy and Storage of Pterocarya Fraxinifolia (Juglandaceae) Seeds: Application in Hyrcanian and Colchis Forest Conservation

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Desiccation, Dormancy and Storage of Pterocarya Fraxinifolia (Juglandaceae) Seeds: Application in Hyrcanian and Colchis Forest Conservation Canadian Journal of Forest Research Desiccation, dormancy and storage of Pterocarya fraxinifolia (Juglandaceae) seeds: application in Hyrcanian and Colchis Forest conservation Journal: Canadian Journal of Forest Research Manuscript ID cjfr-2018-0519.R2 Manuscript Type: Article Date Submitted by the 09-Sep-2019 Author: Complete List of Authors: Wawrzyniak, Mikolaj; Polska Akademia Nauk Instytut Dendrologii w Korniku, Reproductive Biology and Population Genetics Jasińska, Anna; Polska Akademia Nauk Instytut Dendrologii w Korniku, LaboratoryDraft of Systematics and Geography Chmielarz, Pawel; Polish Academy of Sciences Kozlowski, Gregor; Universite de Fribourg, Department of Biology; Museum Fribourg, Natural History biodiversity, Georgia, critical moisture content, cryopreservation, seed Keyword: banking Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/cjfr-pubs Page 1 of 29 Canadian Journal of Forest Research 1 Desiccation, dormancy and storage of Pterocarya fraxinifolia (Juglandaceae) seeds: 2 application in Hyrcanian and Colchis Forest conservation 3 Mikołaj Wawrzyniak1,*, Anna Katarzyna Jasińska2, Paweł Chmielarz1, Gregor Kozlowski3,4 4 1Department of Reproduction Biology and Population Genetics, Institute of Dendrology, Polish 5 Academy of Sciences, Parkowa 5, 62-035, Kórnik, Poland 6 2Laboratory of Systematics and Geography, Institute of Dendrology, Polish Academy of 7 Sciences, Parkowa 5, 62-035, Kórnik, Poland 8 3Department of Biology, University of Fribourg, Chemin du Musée 10, CH-1700 Fribourg, 9 Switzerland 10 4Natural History Museum Fribourg, Chemin du Musée 6, CH-1700 Fribourg, Switzerland 11 *Corresponding author. E-mail address:Draft [email protected] (M. Wawrzyniak) Tel.: 12 +48 61 81 70 033; ORCID: https://orcid.org/0000-0002-4297-5741 13 1 https://mc06.manuscriptcentral.com/cjfr-pubs Canadian Journal of Forest Research Page 2 of 29 14 Abstract 15 Pterocarya fraxinifolia (Juglandaceae) is a model relict tree species native to South Caucasus 16 and it is a typical element of threatened riparian forests. Intensive land transformations which 17 is common in Transcaucasia, resulted in loss of natural habitat and population decline of the 18 species. One of the method for ex situ conservation is seed banking. Cryopreservation in liquid 19 nitrogen (LN; -196°C) is particularly of interest as it allows safe preservation of valuable plant 20 genetic resources. However, the feasibility of seed cryopreservation is related to their 21 desiccation tolerance and intrinsic composition. In this study, we examined the physiological 22 P. fraxinifolia seed traits from which desiccation tolerance is unknown or controversial and 23 their feasibility to cryopreservation,. Additionally, we tested stratification methods for 24 dormancy assessment. Results showedDraft that seeds survived desiccation to a 2.8% seed moisture 25 content (MC) and germinated in 64% . Stratification at temperature 3°C for 8 weeks was proven 26 to be both fast and effective. For cryopreservation, seed MC ranging between 2.8% and 18.1% 27 was determined to be safe. There was no difference in seedling emergence in seeds stored for 28 one year regardless of the storage temperature (-3°, -18° or -196°C). Based on our results, P. 29 fraxinifolia seeds can be classified as orthodox. This study demonstrates for the first time the 30 feasibility of cryopreserving P. fraxinifolia seeds. 31 Keywords: seed banking, cryopreservation, biodiversity, critical moisture content, Georgia 32 2 https://mc06.manuscriptcentral.com/cjfr-pubs Page 3 of 29 Canadian Journal of Forest Research 33 Introduction 34 The conservation of plant genetic resources is an established and globally recognized priority 35 because the variability of species is fundamental to forestalling their extinction. It is estimated 36 that there are more than 300,000 species of land plants, all of which are essential for the proper 37 functioning of local ecosystems (Corlett 2016). However, many populations of plants are 38 currently threatened by loss, fragmentation and degradation of habitats as well as 39 overexploitation, invasive species and climate change (Groom et al. 2005). Conservation 40 programs for threatened species can follow a variety of criteria, i.e., economic value, ecological 41 value or species uniqueness. One of the unique and irreplaceable parts of the plant kingdom is 42 relict trees. These surviving remnants have been present on the Earth for millions of years, 43 representing evolutionary processes andDraft outlasting environmental, biogeographical and climate 44 changes. Despite their continued existence over millions of years, they usually retain a narrow 45 distribution in so-called refugia (Maharramova et al. 2017; Kozlowski et al. 2018) such as 46 Hyrcanian area in the North Iran. The unique ecosystems of Hyrcanian Forests along with plains 47 around the Black Sea (Colchis Forests) are one of the most valuable biosphere genetic resources 48 in the World, which are a shelter of many relict species such as: Parrotia persica (DC.) C. A. 49 Mey., Gleditsia caspica Dest., Zelkova carpinifolia (Pall.) K. Koch. and Pterocarya fraxinifolia 50 (Poir.) Spach. (Naqinezhad et al. 2018). 51 The possibility of in situ conservation has its limitations. A holistic approach to 52 conservation is often difficult to fund and maintain (Diamond 1989), therefore, ex situ 53 conservation, such as seed banking, seem to be most suitable for seed bearing species, as it 54 provides inexpensive and effective long time strategy (Bewley et al. 2013; Zaritzky 2015). The 55 viability of stored seeds depends on the seed moisture content and storage temperature 56 (Vertucci and Roos 1990). Seed storability at low temperature depends on its desiccation 57 tolerance, as access water can crystallize and cause intracellular damage (Wesley-Smith et al. 3 https://mc06.manuscriptcentral.com/cjfr-pubs Canadian Journal of Forest Research Page 4 of 29 58 2014). The practical categorization proposed by Roberts (1973) and then supplemented by Ellis 59 et al. (1990) considers three categories of seeds divided on the basis of their desiccation 60 sensitivity: orthodox (seeds tolerate desiccation below a 5% moisture content, MC, fresh- 61 weight basis), recalcitrant (seeds that do not tolerate desiccation), and intermediate (seeds that 62 exhibits characteristic of both former categories i.e. seeds that tolerate moderate desiccation but 63 lose viability during subsequent storage at subzero temperatures). However, careful study of 64 seeds physiology is important; seeds storage behavior is often more complex as cellular 65 response on water and temperature thresholds create more in-between responses (Walters 2015). 66 Cryopreservation in liquid nitrogen (LN, between -130 and -196 °C) represents a safe 67 and cost-effective option for the long-term storage of germplasm. In recent years, 68 cryopreservation has become a successful method for conserving unique (i.e., endemic, 69 endangered, and economically valuable)Draft species, both as vegetative tissues such as dormant 70 buds and shoot tips and generative organs such as seeds and embryos (Engelmann 2011; 71 Lambardi and Shaarawi 2017). For orthodox seed species, cryopreservation is an alternative to 72 traditional storage at -20 °C and is of particular importance for long-term storage (10 to 100s 73 of years; Reed 2008), and it is the only method for the long-term storage of intermediate and 74 recalcitrant seed species. Cryopreservation of the seeds of woody species has proved to be 75 effective in both orthodox and recalcitrant seeds in many tree species, such as Alnus glutinosa, 76 (Chmielarz , 2010a, ), Coffea spp. (Dussert et al. 1998), Prunus armeniaca (Malik and 77 Chaudhury 2010), Prunus padus (Popova et al. 2016) and others. Together with the ex situ 78 approach, cryopreservation has been successfully utilized in conservation programs for 79 herbaceous plants, such as Cosmos atrosanguinea (Wilkinson 2002) or Lomandra sonderi 80 (Menon et al. 2014). Cryopreservation together with modern biotechnology has been 81 implemented in the conservation programs of many national floras, such as those of Brazil 4 https://mc06.manuscriptcentral.com/cjfr-pubs Page 5 of 29 Canadian Journal of Forest Research 82 (Pilatti et al. 2011), Spain (González-Benito and Martín 2011), Australia (Ashmore et al. 2011) 83 and others. 84 In our study, we examined Caucasian wingnut (Pterocarya fraxinifolia (Poir.) Spach.) 85 of the walnut family (Juglandaceae) as a model relict tree species which belongs to the so-called 86 Arcto-Tertiary relict trees and grows in the lowlands and riparian habitats of the Southern 87 Caucasus, Anatolia and Caspian (Hyrcanian) forests of Iran, Georgia, Turkey and Azerbaijan 88 (Boratyński and Boratyńska 1975; Kozlowski et al. 2018). During the Oligocene and Miocene, 89 the species was widespread in the Northern Hemisphere, but due to climate cooling over the 90 last 15 million years, it became restricted to southern refugial areas characterized by a more 91 stable climate (Manchester 1987; Milne and Abbott 2002; Maharramova et al. 2017). In some 92 locations, the population growth of the species depends on individual clonal reproduction via 93 root sprouts, as natural regeneration viaDraft seeds is poor, and a majority of the seeds is empty (up 94 to 90%; Yilmaz 2015). Its populations are characterized by intermediate to low genetic diversity 95 (Akhani and Salimian 2003;
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